Osteoarthritis is a debilitating condition associated with cartilage and joint dysfunction caused by trauma or aging that severely affects patients' quality of life resulting in a yearly burden of approximately 15 billion dollars on US healthcare. Cartilage regeneration is inherently inefficient and remains an unmet medical need that increases the propensity for development of arthritic conditions. Cell-based therapeutic approaches for repairing focal cartilage defects have utilized autologous adult chondrocytes or adult mesenchymal stem cells (MSC) but with limited success due to generation of inferior fibrocartilage and paucity of cells. An abundant autologous source like human induced pluripotent stem cells (hiPSC) is therefore attractive for engineering cartilage. Additionally, generation of developmentally `younger' chondrocytes from human iPSC akin to the neonatal or juvenile chondrocytes are expected to possess a higher regeneration potential than adult chondrocytes. The aim of this research proposal is therefore to generate iPSC and MSC from the same healthy or OA donor and comparing their potential for cartilage regeneration in vitro and in an osteochondral defect in a rat model. Towards this end, a major advance will be the generation of footprint-free human iPSC i.e. without the use of viral vectors that permanently integrate into the genome. Use of synthetic mRNA will allow generation of safe and clinically relevant hiPSC. Additionally, we have recently developed an efficient methodology to direct human iPSC (hiPSC) differentiation towards chondrocytes (or chondroprogenitor cells) using transient exposure to a series of growth factors. We will define molecular and functional characteristics of hiPSC- and hMSC-derived chondrocytes to relate with their functional capabilities. Secondly, we will optimize a biomimetic hydrogel scaffold for the maturation and implantation of human iPSC- and hMSC-derived chondrocytes. Thirdly, the potential of the human iPSC- and hMSC-derived chondrocytes to repair a focal cartilage defect will be tested in a rat model of surgically induced osteochondral defect along with long-term safety studies in mice. Collectively, these studies will help evaluate and provide a mechanistic understanding of whether a hiPSC-based cellular therapy will be superior to hMSC-based therapy and successful completion can provide the impetus to further develop a clinically applicable iPSC- based therapy for focal cartilage injury.